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Vortex gently.
Cells were fixed with cold 70% ethanol (vortex gently, incubate for 30 min on ice), centrifuged (10 min, 500 g, room temperature), and the pellets vortexed gently and re-suspended in 1 ml 2N HCl/0.5% Triton X-100 (30 min incubation at room temperature) to denature the DNA.
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After vortexed, 445 μL of acetonitrile was added and again vortexed gently for 2 min, then centrifuged for 8 min at 12500 rpm at 4°C.
The PEI-modified TPGS-b- PCL-ran-PGA) nanoparTPGS-b- PCL-ran-PGATPGS-b- PCL-ran-PGAlasmid DNA soluTPGS-b- PCL-ran-PGA/P raTPGS-b- PCL-ran-PGAenanoparticle
The PEI-modified TPGS-b- PCL-ran-PGA) NP soluTPGS-b- PCL-ran-PGA TPGS-b- PCL-ran-PGAsoluTPGS-b- PCL-ran-PGA/P raTPGS-b- PCL-ran-PGAeNPly.
The pellet was washed three times with 200 µl of wash buffer, 50 µl of elution buffer was added, and the suspension was vortexed gently for 30 s and pelleted by centrifugation.
Specifically, 20 µg glycogen (or 5 µl glycoblue, Ambion), 0.6 volumes 100%isopropanol and 0.1 volumes 3M Sodium acetate pH 5.2 were added, the mixture was immediately vortexed gently, and centrifuged at room temperature at maximum speed for 25 minutes to pellet the nucleic acids.
The sample was vortexed gently and incubated at 60°C for 30 min followed by centrifugation.
Tubes were then vortexed gently, and reverse transcription was performed in Veriti® Thermal Cycler (Applied Biosystems, Foster City, CA, USA).
The samples were then vortexed gently for 15 min followed by a 10 min centrifugation at 3000 rpm to separate the aqueous and organic layers.
Then 90 μL 50 mM ammonium bicarbonate were added to the membrane and the filter was vortexed gently to re-suspend de-salted proteins.
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